A. actinomycetemcomitans, serotype b has attracted much interest during the past three decades due to its close association with periodontitis in young individuals and the enhanced expression of a leukotoxin (LtxA). A typical feature of this genotype is a 530-base pair (bp) deletion in the ltxCABD promoter region controlling leukotoxin expression. In the present work, we have characterized serotype b strains with four additional promoter types. Two novel types have been recognized, that is, one with a 230-bp deletion and one with a 172-bp duplication. Moreover, a strain with a 640-bp deletion and three strains with a full-length promoter, including the type strain Y4, were included in the present study. The seven strains were characterized by multi locus sequence typing (MLST) and arbitrarily primed polymerase chain reaction (PCR) and assessed for LtxA production. MLST showed that the strains with the non-JP2-like deletions represented distinct monophyletic groups, whereas the JP2 strain, HK1651, represented a separate branch. LtxA production was high in all three strains with a promoter deletion, whereas the other four strains showed significantly lower levels. It can be concluded that the genetic characterization and determination of LtxA production of A. actinomycetemcomitans isolates from individuals with periodontitis can contribute to the identification of novel virulent genotypes of this bacterium. The JP2 genotype of  Aggregatibacter actinomycetemcomitans is associated with periodontitis in young people [The toxin-producing bacterium g people ,2,3,4. Tg people . Since tg people ,7. The ag people . The leug people .ltxCABD gene operon encoding the leukotoxin (LtxA) is controlled by one promoter, referred to as the leukotoxin promoter [The promoter . It has promoter . This gepromoter ,15,16,17cagE gene locus. This gene sequence has been suggested as a suitable diagnostic marker for carriers of highly virulent A. actinomycetemcomitans serotype b and which are at increased risk of developing an aggressive form of periodontitis and appears not to be present in any of the other serotypes [The JP2 genotype produces increased amounts of the leukotoxin (LtxA) and strains of this genotype have been demonstrated to be highly leukotoxic . Individerotypes ,24.Whereas AP-PCR typing mirrors genetic variability within bacterial species without catching specific genes, multilocus sequence typing (MLST) is used for the characterization of bacteria in terms of pathogenicity and dissemination by sequencing specific so-called housekeeping genes ,25. MLSTA. actinomycetemcomitans have been reported, one lacking 640 bp instead of the typical 530 bp and one promoter that carries an 886-bp insertion sequence (IS), referred to as IS1301 [ltxCABD promoter types of A. actinomycetemcomitans serotype b, being one, which contains a 172-bp duplication, whereas the other lacks 230 bp and therefore has a deletion. These newly discovered promoter types have been characterized in comparison with the other well-known leukotoxin promoter types regarding AP-PCR and cagE genotype, MLST phylogenetic analysis and leukotoxicity.In addition to the most common variant with a full-length leukotoxin promoter and the JP2 genotype, two additional promoter variants of s IS1301 ,27. In tSamples from periodontal pockets of patients have been routinely analyzed for the presence of periodontitis–associated bacterial species at the Clinical laboratory at Oral Microbiology, Dental School in Umeå, Sweden, for more than 35 years, according to established methods ,24.A. actinomycetemcomitans-specific medium [2, the total number of viable bacteria and the proportion of A. actinomycetemcomitans in the samples were calculated. Catalase-positive colonies with star-like inner structure were serotyped and leukotoxin promoter typed as described in The samples were collected from periodontal pockets with paper points and sent to the laboratory in an anaerobic transport medium (VMGAIII) . After sc medium . After iA. actinomycetemcomitans strains 581-18U, 582-18U and 046-19U were isolated from subgingival plaque using the procedures described above. Five additional serotype b strains were included in this work for comparison purposes. Three of the strains exhibit a full-length leukotoxin promoter—575G was sampled from a Ghanaian cohort of adolescents (1110-bp promoter) [A. actinomycetemcomitans strains previously reported on by Professor Sirkka Asikainen (1110 bp). Y4  [2 at 37 °C.romoter) . S23A be1107 bp) ,31 is a 1107 bp)  is JP2 g1107 bp) . For expA. actinomycetemcomitans colonies in water. A. actinomycetemcomitans genomic DNA to be used in AP-PCR and whole-genome sequencing was isolated using the GenElute™ Bacterial Genomic DNA kit , following the manufacturer’s instructions. PCR reaction mixtures were prepared using illustra™ PuReTaq™ Ready-To-Go™PCR beads , whereas a KAPA SYBR®FAST qPCR kit  was used for qPCR. The AP-PCR type was analyzed as described earlier [cagE gene was amplified by qPCR as a 623-bp internal fragment using the primers cagE_F2 and cagE_R2, as described previously [DNA templates for conventional PCR and quantitative PCR (qPCR) analysis were obtained by boiling a loopful of fresh  earlier ,23, usin earlier . The str earlier ,24). Theeviously ,35,36. Dhttp://www.ebi.ac.uk/ena).Genomic DNA samples from strains 046-19U, 582-18U and Y4 were prepared as described above and sent to MicrobesNG  for whole-genome sequencing. The obtained trimmed reads were annotated with Rapid Annotation using Subsystem Technology (RAST) (rast.nmpdr.org). The genome of the serotype b strain HK1651 (CP007502) was used as the reference. The whole genome sequences of 456-13U and 575G  and S23ArecA (GenBank accession EF142768), encoding the RecA protein; adk (EF142164), encoding adenylate kinase; frdB (EF142336), encoding fumarate reductase; atpG (EF142218), encoding the gamma subunit of ATP synthase F1; pgi (EF142653), encoding glucose-6-phosphate isomerase; and mdh (EF142568), encoding malate dehydrogenase. In addition, two fragments of the hemoglobin-binding protein pseudogene hbpA (EF142408 and EF142489) and one fragment of the transferrin-binding protein pseudogene tbpA (EF142817) were selected. Nucleotide sequences were obtained either by sequence analysis of PCR fragments or extracted from whole-genome sequencing data. The oligonucleotides and PCR amplification conditions used have been described previously [A. actinomycetemcomitans strains used in the present work was conducted, based on concatenated sequences in the order adk, atpG, frdB, mdh, pgi, recA, hbpA-1, hbpA-2 and tbpA. Evolutionary analyses were conducted in MEGA X  (https://www.megasoftware.net/) [The gene fragments used in MLST were selected previously, based on the sequences of strain HK1651 . Parts o mdh EF1468, encodre.net/) ,38. Evolre.net/) . A total10 bacterial cells/mL (OD600nm = 10.0) by a rocking movement for 1 h in a 0.45% sodium chloride solution in PBS at around 5 °C. The killing assay was based on the reduction of neutral red uptake in LtxA-exposed THP-1 cells stained with this dye. The concentration of THP-1 cells (106) was determined in a hemocytometer before they were added to a 96 welled cell culture plate. Cellular neutral red uptake was quantified by a spectrophotometer and expressed as a percent of a plane control, as described previously [50), the dose that kills 50% of the target cells, was determined in leukotoxin-exposed THP-1 cultures. The target cells were exposed for 2 h to leukotoxic extracts from different concentration of the various A. actinomycetemcomitans isolates or to purified LtxA. The concentration (multiplicity of infection (MOI)) that reduced viability in the THP-1 cultures with 50% determined the LD50 value. LtxA-induced release of IL-1β from the target cells was measured with an enzyme-linked immunosorbent assay (ELISA) kit in accordance to the manufacturer’s protocol .The leukotoxicity, that is, the capacity of the strains to kill THP1-cells and induce IL-1β release from them, was analyzed. LtxA was extracted from suspensions of 10eviously . The ass9 bacterial cells/mL (OD600nm = 2.0) of plate-cultivated isolates were disintegrated by boiling for 5 min together with 1% SDS (1:1). The samples were loaded in a Miniblotter device  with a nitrocellulose (NC) membrane. The NC membrane was treated stepwise with rabbit–anti LtxA [TM XRS + System . Data were expressed as a percent (%) of the banding intensity for the JP2 genotype, HK651. For the determination of the LtxA production with checkerboard (CB), a Western blot-based method was used. A suspension of 2 × 10nti LtxA  and goatEthical clearance for the present study was obtained from the Noguchi Memorial Institute for Medical Research, University of Ghana (IRB 000 1276) and from the local ethical committee of Umeå University, Sweden (Dnr 2010-188-31M). p-values. The 95% confidence interval (p ≤ 0.05) was used for the indication of significant differences from the JP2 genotype of A. actinomycetemcomitans (HK1651).The statistical significance of the leukotoxicity data was calculated with ANOVA and a post-hoc test with Bonferroni correction to determine the A. actinomycetemcomitans in our clinical laboratory of the Dental School at Umeå, Sweden. One was detected in two periodontal plaque samples collected from a 21-year-old female patient of Ethiopian descent and with severe periodontal lesions located on the first molar. These isolates were denoted 581-18U and 582-18U (A. actinomycetemcomitans (A. actinomycetemcomitans (isolate denoted 046-19U) (We recently identified two novel types of leukotoxin promoter of  582-18U . The samcomitans . The sec046-19U) . It was The isolates were confirmed to be of serotype b according to PCR analysis (data not shown); however, deviant patterns were observed when the leukotoxin promoter regions of the isolates were amplified by PCR . DNA seqAP-PCR characterization of the isolates showed that the strain with the duplication (582-18) was an AP-PCR type 1, whereas the novel deletion type, strain 046-19U, was found to be an AP-PCR “other” type . cagE gene locus, in contrast to 046-19U, which was found to be cagE-negative  observed in these gene fragments, six MLST sequence types could be identified, denoted 1‒6. No variation among the strains were observed in the adk, mdh and recA gene fragments. Strain Y4 was the only one to have a SNP in the frdB fragment and the hbpA1-fragment was shorter in strains 046-19U and Y4, containing only nucleotide positions 370‒439  A. The LDisolates . A. actinomycetemcomitans isolates also induced a higher secretion of IL-1β from the exposed THP-1 cells than the four other isolates  promoter structure are generally considered weakly leukotoxic. However, a subset of serotype b strains with full-length promoters can exhibit high leukotoxicity. Properties in common for them are that they share the same AP-PCR type, that is, AP-PCR type 1 [cagE gene locus [A. actinomycetemcomitans strains with specific genetic rearrangements within the promoter, both deletions and insertions, have been reported to be highly leukotoxic [R type 1  and thatne locus ,24. A. aukotoxic ,42. In tFor studying LtxA expression, various methods have been used over several decades ,44,45,46A. actinomycetemcomitans with a complete leukotoxin promoter has been suggested to be due to the fact that a transcriptional repressor, hitherto unknown, can bind to a region in the promoter (bp 523–1053), which is not present in JP2 genotype strains due to their 530-bp deletion [The generally low production of LtxA by deletion . Indeed,deletion . As expeA. actinomycetemcomitans strain AaIS1, which has an 886-bp IS1301 insertion in the ltxCABD promoter [AaIS1 produces enhanced amounts of LtxA has been proposed to be that IS1301 displaces an upstream negative cis-acting DNA sequence [IS1. The reason for this discrepancy is not known. However, interestingly, by being deprived of bp 44-264 the promoter of strain 046-19U actually contains the region where the potential repressor might bind. This suggests that other regions of the promoter may also be targeted by regulatory protein(s). This would be consistent with observations of the promoter . That prsequence . To the A. actinomycetemcomitans, which indicates that the 230-bp leukotoxin promoter type is interestingly divergent in more than one aspect. However, like the other non-AP-PCR 1 types, it does not carry cagE. Like the leukotoxic activity, the AP-PCR pattern varied among the tested strains. Among them, four are AP-PCR type 1 . This AP-PCR type has been found to be more associated with enhanced leukotoxicity than the other AP-PCR-types ,23. AmontpbA gene fragment. In addition, Y4 carries an SNP (T > C) at position 131 within the frdB gene. None of these SNPs could be observed in any of the 66 JP2 strains studied earlier [When the genetic variability of the strains was studied by MLST, six sequence types were recognized based on the identification of eight SNPs. Although the numbers of SNPs differed substantially between the strains, their genetic distance was relatively low, as shown in the phylogenetic tree. Among the four genetic subgroups identified by the MLST in the present work, the JP2 genotype strain, HK1651 was represented in a separate branch. MLST analysis of 66 JP2 strains has revealed that they are closely related genetically . Interes earlier .A. actinomycetemcomitans that we have focused on in the present study, the one with the complete promoter type (≈1106–1112 bp) is without doubt the most common . Among these individuals, 693 carried A. actinomycetemcomitans with deleted or extended leukotoxin promoters is very low. There are reasons to believe that the prevalence could be substantially underestimated due to the absence of sampling and leukotoxin promoter typing, especially regarding the other serotypes. This also means that access to valuable laboratory diagnostics parameters for treatment of periodontal diseases is limited, as well as for studies of the geographic dissemination of A. actinomycetemcomitans with various leukotoxin promoter structure.Overall, it seems that the prevalence of Aggregatibacter actinomycetemcomitans has extended the concept of leukotoxin promoter structure versus leukotoxicity, genetic diversity and geographic dissemination of this bacterium. The identification of two additional leukotoxin promoter types of


To develop population norms for the EQ-5D-5L questionnaire based on a representative sample of Moscow citizens.We used quota sampling accounting for sex, age group and administrative district of residence. Respondents in randomly selected outdoor and indoor locations were surveyed with the official Russian paper-and-pencil version of the EQ-5D-5L questionnaire and a set of socio-demographic questions. We estimated four types of EQ-5D results: the distribution of limitations according to EQ-5D-5L dimensions, the perception of the health-related quality of life (HRQoL) with a visual analogue scale (EQ VAS), the unweighted score for a respondent’s health state  and the Russian health preferences-based weighted score (EQ index). In order to estimate the EQ-5D-5L index, we used a newly developed Russian EQ-5D-3L value set, together with EuroQol Group cross-over methodology.A total of 1020 respondents (18–93 years old) from the general Moscow adult population completed the EQ-5D-5L questionnaire. HRQoL domains with the largest number of identified health limitations were pain/discomfort (48.6%) and anxiety/depression (44.1%). Two hundred seventy-nine respondents (27.0%) did not report any health restrictions. The mean EQ VAS and EQ-5D-5L index were 74.1 (SD 17.3) and 0.907 (0.106) respectively. Multivariate analysis showed that female sex, advanced age and lack of access to the Internet had a negative influence on HRQoL, whereas residence in certain districts had a positive impact.The study provides population norms of health-related quality of life in Moscow, measured according to the EQ-5D-5L questionnaire. These reference values can be used to optimise the effectiveness of resource allocation in healthcare.The online version of this article (10.1007/s11136-020-02705-0) contains supplementary material, which is available to authorised users. Over the past few years, health technology assessment (HTA) has been implemented in most Central and Eastern European (CEE) countries—based on international standards or through developments to the original HTA system, as in post-Soviet countries . Russia,When measuring HRQoL, one can choose between generic and disease-specific instruments . GenericPopulation reference data for a specific country or region , can be used to compare profiles for patients with particular conditions with data for the average person in the general population from a similar age and gender group. Additionally, population norms enable comparison of the burden of the disease with the general population’s health, as well as the measurement of health inequalities .Moscow has one of the largest municipal economies in Europe, which also accounts for more than one-fifth of the country’s gross domestic product. In Russia, Muscovites are in second place in terms of life expectancy, second only to inhabitants of the Republic of Ingushetia . AlthougOur study aimed to develop population norms for HRQoL in Moscow, based on the EQ-5D-5L questionnaire. Bearing in mind the size of Russia, and knowing that most of the pilot projects are carried out firstly in Moscow before encompassing other regions, we have decided to start from the capital.okrug). The official division of Moscow in place until 2012, with ten administrative areas, was used. Seven age strata were distinguished . Quotas were built based on the Russian Statistics Office (ROSSTAT) data from 2014 [To make our study representative, we used quota sampling. Quotas accounted for sex, age group and the district of residence  and a visual analogue scale (EQ VAS). The descriptive system includes five dimensions: mobility (MO), self-care (SC), usual activities (UA), pain/discomfort (PD), and anxiety/depression (AD). EQ-5D-5L dimensions comprise five levels of severity: no problems, slight problems, moderate problems, severe problems, and extreme problems . ResponsThe additional socio-demographic questionnaire included questions about: respondents’ age, level of education, monthly income, smoker/non-smoker and Internet access and usage. The question about the education provided nine possible answers. For data analysis, the responses were aggregated into three levels: primary (up to 9 years of education), secondary  or higher education .Two researchers  approached randomly selected respondents in randomly chosen locations, including parks, bus stops, transport hubs, car washes, malls, markets, restaurants, cafes and universities. The respondents were asked to fill in the EQ-5D-5L on their own but were guided by the interviewer if necessary. Answers to questions concerning demographic characteristics were collected by the interviewers. Items were read by the interviewer a maximum three times, and no additional comments were provided. A total of 10% of the interviews were subjected to data input quality control (the data of every tenth questionnaire in the paper form were verified with the data entered in the electronic database). Interviewers’ cross-validation was performed (MH carried out control of the data from the questionnaires entered by AT and vice versa).We estimated four types of EQ-5D results: (1) the distribution of limitations according to EQ-5D-5L dimensions, (2) the perception of the health-related quality of life with EQ VAS, (3) the unweighted score for the respondent’s health state—the Level Sum Score (LSS), and (4) the weighted score for the health state—the EQ-5D-5L index. For continuous variables, we calculated the following descriptive statistics: mean, standard deviation and 95% confidence interval. Estimations were presented for the whole sample, as well as for the seven predefined age groups, in the EuroQol Group’s standardised format, to facilitate comparative research. We used multiple linear regression to examine the associations of socio-demographic characteristics with the EQ-5D-5L index and EQ VAS scores. All variables, including age, were entered into the models as categorical variables. Regression coefficients were presented together with information about the level of statistical significance. The analysis was carried out using Microsoft Excel and StatsDirect software, version 2.8.0 .From February to October 2017, a total of 1020 respondents from Moscow completed the EQ-5D-5L questionnaire. The sample approximated to the general adult Moscow population in terms of age, gender and district of residence . The most striking difference concerned the youngest age group (18–24 years), and the domains pain/discomfort and anxiety/depression, where the absolute difference between the frequency of problems reported by women and men was 33.5% and 32.4%, respectively .In total, we observed 170 different health states (out of the 3125 possible defined by EQ-5D-5L). The five most common health states accounted for 50.7% of cases, while the ten most common accounted for 60.8% of cases . As in other general population studies, the health-related quality of life of Moscow residents measured according to the EQ-5D-5L index or LSS, clustered around the highest values, whereas when measured by EQ VAS it was far from the top.Detailed characteristics of the EQ-5D-5L index and EQ VAS, according to gender and age group, are shown in Table In general, lower HRQoL values start to be evident from the age of 45, and the decline in HRQoL accelerates from 65 years old. Women are characterised by worse HRQoL ratings than men, except for two age groups  in EQ VAS.Electronic Supplementary Material  presents the descriptive analysis of population data, preceding the regression analysis. Better educated respondents tend to report fewer limitations within UA, but more within AD. Young (18–24 years) males with primary or secondary education reported fewer problems within all EQ-5D-5L dimensions, than those with higher education. Young (18–24 years) females with primary or secondary education reported substantially fewer problems within PD and AD dimensions , than those with higher education. Better educated women had higher EQ VAS and EQ-5D-5L index scores across all age groups. Relationship between current smoking and HRQoL was unsystematic. Higher income was associated with better EQ VAS and EQ-5D-5L index scores, fewer problems within MO, UA and PD dimensions, but more anxiety and depression.Table To the best of our knowledge, this is the first study to estimate population norms for the descriptive part of the EQ-5D-5L questionnaire, EQ VAS, EQ-5D-5L index and Level Sum Score, among a representative sample of Moscow inhabitants. Norms for the EQ-5D-5L index were obtained through the usage of a recently developed Russian EQ-5D-3L value set and EuroQol Group cross-over methodology. Moscow’s normative data may be used as reference values in future EQ-5D-based studies. We have found that HRQoL is worse in women than in men across almost all age groups, especially in the youngest one (18–24 years).One of the limitations of our study may be the moderate sample size (about 1000 respondents), which is clearly smaller than, for example, that used in a Spanish study  , 15. We Although since July 2012 there has existed a new official administrative division of Moscow (two new districts were added), we decided to limit our study to Moscow’s former division into 10 zones. The two new areas have a suburban character, are distant from the city centre (up to 80 km), less populated, have limited access to the metro system, and the health-related quality of life of people living there could be different than for other Muscovites.Our sample proved to be representative for the Moscow population in terms of sex, age group and the district of residence. However, we based our study on quota sampling, which is a less optimal solution than the random sampling used in other Central and Eastern European studies, such as in Slovenia or Poland  , 26.Although accessibility of the Moscow transportation system for the disabled has improved over the last few years, since the Passenger Mobility Centre was opened in 2013, limitations for handicapped people on Moscow transportation systems remains significant, and we were not able to interview any person with a visible disability. According to official statistics, disabled people constitute about 8.5% of the Russian population . One mayOur sample was not perfectly balanced in terms of education. Higher education constitutes 42% in Moscow reference data and 59% in our study group, primary education—6.5% and 0.5%, respectively. In general, the level of education of Muscovites seems high compared to other populations. Our sample was additionally skewed in the direction of higher levels of education. This could be a particular limitation in situations in which Moscow population norms will be used to evaluate health care programmes that affect the less-educated set of beneficiaries, for example, citizens of other Russian regions.A high percentage of respondents (about 22%) refused to answer the question regarding income. As stated previously, refusal to answer among Russian respondents, particularly in Moscow, is frequently of significant proportions, partly due to high levels of distrust towards strangers .n = 545) [n = 1190) [n = 1800) [There is a long tradition of health surveys carried out among the Moscow population. In the last 3 decades several have taken place: a postal survey in 1991 (n = 545) , the Mos = 1190) , 30, 31  = 1800) , 33. In We did not identify any population norms survey, neither in Moscow nor Russia, based on the EQ-5D-5L questionnaire. However, in general, studies including EQ-5D are quite numerous, especially those undertaken in disease-specific populations and published in the Russian language. The Russian version of EQ-5D-5L has been successfully validated in a group of 163 patients with spondylarthritis . Recent The lower health perceptions among Moscow women was noted in the early nineties in a study comparing them with women from Helsinki , and subOne of the potentially interesting results of our study is the evidence of a particularly low HRQoL in young females, as measured with standardised tools. Among women aged 18–24 years, over 60% reported problems with anxiety/depression and over 53% with pain/discomfort. The assessment of health with EQ VAS was 8.3 points lower than in men from the same age group.Many different factors could lie behind this phenomenon. The Russian sociologist Elena Varshavskaya  writes aHRQoL population norms for Moscow, taking into account the size of the city—over 11.8 million inhabitants, constitute a standalone value. Nevertheless, our study may be treated as a pilot project, preceding a nation-wide survey. Given the geographical extent of the country, the implementation of Russia population norms study can be a challenge but should constitute the next logical stage of research. Current Moscow population norms should be re-estimated, when the directly measured EQ-5D-5L, becomes available.This study presents population norms for the EQ-5D-5L health questionnaire, based on a representative sample of Moscow inhabitants. These reference values, distinguishing age and gender groups, can be used to optimise the effectiveness of resource allocation in healthcare.Supplementary file1 (PDF 424 KB)Below is the link to the electronic supplementary material.


PBT has been reported to be effective against esophageal cancer. However, apart from 5-FU and CDDP, almost no other drug has been tested in combined chemotherapy with PBT. Therefore, we investigated the effects of a poly (ADP-ribose) polymerase inhibitor on enhancing proton beam effects using esophageal cancer cell lines that exhibit resistance to radiation and CDDP. Esophageal squamous cell carcinoma cell lines OE-21 and KYSE-450 were exposed to the drugs for 1 h prior to irradiation. The cell survival curve was obtained using a clonogenic assay and the sensitizing effect ratio (SER) was calculated. The clonogenic assay was used to compare the effect of multi-fractioned irradiation between 8 Gy/1 fraction (fr) and 8 Gy/4 fr. γH2AX, Rad51, BRCA1, BRCA2 and 53BP1 foci were detected via immunofluorescence. Olaparib exhibited an SER of 1.5–1.7 on PBT. The same sensitizing effect was exhibited in multi-fractioned irradiation, and the combined use increased the expression of double-strand breaks and homologous recombination-related genes in an additive manner. Such additive effects were not observed on non-homologous end joining-related genes. We demonstrated that olaparib has a high sensitizing effect on PBT in platinum- and radiation-resistant esophageal cancer cells. Our results suggest a potential clinical application of olaparib-proton irradiation (PT) against platinum- and radiation-resistant esophageal cancer.Proton beam therapy (PBT) combined with chemotherapy, such as  We use the same conditions with other protein detection. First, antibodies for Rad 51 (sc-8349), BRCA1 (sc-642), BRCA2 (sc-28235) and 53BP1 (sc-22760)  were used and the foci in each cell were counted using HCS Studio 2.0 Cell Analysis Client Software, then the mean number of foci per nucleus was calculated. More than 100–300 cells per well were evaluated following irradiation. The procedures were normalized with previously reported protocols. Immunostaining was evaluated for each antibody using a fluorescence microscope. Gamma H2AX was used to assess normality, and it was confirmed that it was radiation dose-dependent .The cells were seeded onto 96-well dishes at a density of 1.0 × 100) were generated using OriginPro version 8.5.1 software (OriginLab Corp).A PARP inhibitor, olaparib (AZD2281), was purchased from Selleck Chemicals . After the initial treatment with olaparib, olaparib was added again each time the medium was changed. In the radiation combination experiment, the drug was added 1 h before irradiation. Clonogenic survival curves and the sensitization enhancement ratio at 10% (SERt-test.The individual experiments were performed at least in triplicate. The statistical significance of the observed differences was analysed using Student’s Sensitivity of esophageal cancer cell lines to anticancer agents and radiation We confirmed that esophageal cancer cell lines OE-21 and KYSE-450 displayed resistance to photon and proton irradiation and 5-FU and CDDP, respectively  and 2. RCDDP is the most commonly used drug for enhancing radiosensitivity in esophageal cancer. The radiosensitizing effect of olaparib was compared with that of CDDP in OE-21 and KYSE-450 cells after proton irradiation with a 2-Gy dose. Next, we compared the colony-forming abilities between the control group and the drug-only group after radiation. We found that KYSE-450 cells exhibited a higher resistance to cytotoxic drug and radiation alone which are used as clinical treatment for esophageal cancer . In OE-2Next, we investigated the proton-sensitizing effect of olaparib in detail using a clonogenic assay. Olaparib exhibited a SER of 1.7 for OE-21 cells and 1.5 for KYSE-450 cells that had high radiation and CDDP resistance . The effTo investigate olaparib and proton-induced DNA damage and repair of esophageal cancer cells, DNA damage after the treatment and expression of a DNA repair enzyme were examined. We used the number of γ-H2AX foci as an indicator of DNA damage. 53BP1 was used as a NHEJ reporter, and BRCA1, BRCA2 and Rad51 were used as HR markers , 15. BotIn addition, we evaluated the intensity of γ-H2AX foci, which is an indicator used for DNA damage assessment and to count the number of foci  16, 17], 1716, 1The levels of BRCA1, BRCA2 and Rad51 were significantly higher in the olaparib-PT group than in the control and olaparib-only groups. In contrast, proton irradiation did not increase the 53BP1 expression level .In the present study, we observed that esophageal cancer cells resistant to radiation and DNA-damaging agents were sensitive to the PARP inhibitor olaparib. We examined the sensitivity to radiation alone and to the drug alone in two esophageal cancer cell lines: OE-21 and KYSE-450. In accordance with previous reports, OE-21 cells were moderately affected whereas KYSE-450 cells were highly affected by both treatments. KYSE-450 cells also displayed resistance to 5-FU and CDDP, two commonly used radiosensitizers. NHEJ is the primary pathway for repairing DNA damage caused by agents such as 5-FU and CDDP, suggesting that these cells are resistant to general DNA damage .The cells were also resistant to radiation. Remarkably, the similar sensitivity displayed by OE-21 and KYSE-450 cells to olaparib was not consistent with their RT-resistance.We determined that olaparib has an excellent proton radiosensitizing effect in radiation and CDDP-resistant esophageal cancer cells.The radiosensitizing effect of olaparib was compared with that of CDDP in OE-21 and KYSE-450 cells . CDDP anOur results are consistent with previous reports of RT-resistant cell lines being immune to DNA-damaging agents such as CDDP. In contrast, olaparib caused increased proton radiosensitization even in RT-resistant cell lines. Although both HR and NHEJ are important for DNA repair in response to CDDP alone, radiation alone or radiation combined with CDDP, NHEJ is believed to be the primary pathway , 21. ConInterestingly, it has recently been reported that olaparib may be more effective in cancer cells with high NHEJ activity. The formation of a complex containing 53BP1 and Rev7 on DSBs typically activates NHEJ. However, a recent report suggests that a mutation of the Rev7 gene reduces NHEJ activity and stimulates HR . Thus, iFurthermore, the current standard treatment for esophageal cancer is a combined platinum and radiation therapy with multifractionated radiotherapy involving a single dose of 1.8–2.0 Gy. Our study confirmed that even multidose irradiation of 2 Gy, which mimics actual esophageal cancer treatment, exhibits a high proton sensitization effect. Sensitizers in PBT have not been largely reported thus far, and the present study verifies for the first time a sensitization effect using 2 Gy multifractionated radiotherapy.To evaluate the biological effect of olaparib and proton irradiation, we assessed the dynamics of γ-H2AX foci, intensity and expression of DNA repair genes. DNA damage was observed to be increased in the proton-only and olaparib-only groups, and an additive increase in the olaparib and proton combination group was also observed .Notably, in the olaparib-PT group of OE-21 cells, there was no significant increase in the number of foci compared with the olaparib-only group, but there was a prominent increase in the intensity of the foci. Although the number of γ-H2AX foci is the most common indicator of DNA damage in radiation biology, the intensity of γ-H2AX foci is also frequently used , 16. BecIn our study, we detected higher expression levels of three HR repair enzymes, i.e. Rad51, BRCA1 and BRCA2, in both the proton-only and olaparib-only groups . FurtherHowever, olaparib has been reported to exhibit a high sensitizing effect on photon therapy with an SER of 1.5–2.1 where there are no OCDLs . FurtherRadiation causes SSBs and DSBs . AlthougThis mechanism suggests that PARP inhibitors may also be effective for cells that are resistant to platinum and photon therapy with high NHEJ activity. Recently, DNA damage drugs and radiation resistant is caused by high NHEJ activity and high copy number alterations can predict radiation resistance . FurtherOlaparib offers a promising alternative because it helps reduce bone marrow suppression or mucosal damage, its dose-limiting toxicity does not overlap with radiation, and its anti-tumor effect is elevated even in hypoxic conditions that usually cause radiation resistance , 18, 24.We demonstrated that olaparib has a high sensitizing effect on PBT in platinum- and radiation-resistant esophageal cancer cells. The sensitizing effect was remarkable in multisplit irradiation, and an additive effect of HR-dependent DSB was considered the mechanism of action. Our findings suggest novel clinical applications of olaparib-PT against platinum and radiation-resistant esophageal cancer.The authors have no conflicts of interest.Fig_S1A_rrz088Click here for additional data file.Fig_S1B_rrz088Click here for additional data file.


Newborn screening for congenital adrenal hyperplasia (CAH) has one of the highest false positive rates of any of the diseases on the Wisconsin panel. This is largely due to the first-tier immune assay cross-reactivity and physiological changes in the concentration of 17-hydroxyprogesterone during the first few days of life. To improve screening for CAH, Wisconsin developed a second-tier assay to quantify four different steroids  by liquid chromatography–tandem mass spectrometry (LC–MSMS) in dried blood spots. From validation studies which included the testing of confirmed CAH patients, Wisconsin established its own reporting algorithm that incorporates steroid concentrations as well as two different ratios—the birth weight and the collection time—to identify babies at risk for CAH. Using the newly developed method and algorithm, the false positive rate for the CAH screening was reduced by 95%. Patients with both classical forms of CAH, salt-wasting and simple virilizing, were identified. This study replicates and expands upon previous work to develop a second-tier LC–MSMS steroid profiling screening assay for CAH. The validation and prospective study results provide evidence for an extensive reporting algorithm that incorporates multiple steroids, birth weight, and collection times. Congenital Adrenal Hyperplasia (CAH) is a group of autosomal recessive disorders caused by enzymatic defects within the biosynthetic pathways of mineralocorticoids, glucocorticoids, and sex hormones. The most common defect, accounting for greater than 90% of CAH cases, is due to 21-hydroxylase (21-OH) deficiency, and has an estimated prevalence in the United States of 1 in 16,000 to 1 in 20,000 . There aNewborn screening for 21-OH deficiency was first introduced in 1977 , with thIn recent years, newborn screening programs have implemented second-tier testing to reduce false positive rates. This second-tier test is typically performed on the original specimen, when the first-tier screening result—elevated 17-OHP—is abnormal. In 2004, Lacey et al. published the first report of a second-tier method for CAH . This meIn 2007, Janzen et al. published an original method validation and prospective study which incorporated analysis of 21-deoxycortisol in dried blood spots, along with the ratio of (17-OHP + 21)/F . The artThis study adds to the literature by outlining the method validation for four different steroids measured by LC–MSMS. Details on the development of an algorithm, which incorporates first-tier testing and second-tier analysis of multiple steroids and associated ratios, stratified by birth weight and the timing of specimen collection, are provided. Lastly, this study summarizes the year-long implementation of the second-tier test and its influence on positive predictive value and false positive screening rates for CAH within the Wisconsin population.n = 144), obtained through the Wisconsin NBS program, were also used for the validation. The true and false positive specimens were stored at −20 °C prior to analysis. For the prospective study, all dried blood spot specimens were collected between 0 and 168 h after birth and were stored at 4 °C after routine NBS was completed.A protocol (2019-0118) for the use of residual dried blood spot (DBS) newborn screening (NBS) specimens was approved by the Health Sciences Institutional Review Board (IRB) at the University of Wisconsin (3/28/2019). For the assay validation, DBS specimens from 18 patients with CAH were obtained from newborn screening laboratories and were de-identified according to the IRB-approved protocol. Residual, known false positives specimens . Plasma and washed RBC were combined in 50/50 v/v (to achieve a 50% hematocrit) to make whole blood.Standards for cortisol (F) and 17-hydroxyprogesterone (17-OHP) were purchased from Sigma . The standard for 21-deoxycortisol (21) was purchased from ChemCruz , and the standard for 4-androstene-3,17-dione (4-A) was obtained from Cambridge Isotopes . The isotopically labeled internal standards D-7, 4-androstene-3,17-dione , D-8, 21-deoxycortisol , D-8, 17-hydroxyprogesterone , and D-4, cortisol  were purchased from CDN isotopes . Acetonitrile and methanol HPLC–MS grade solvents were purchased from J.T. Baker through Fisher Scientific . Grade 903 filter paper for DBS preparation was purchased from Whatman GmbH . Sera Con II charcoal filtered de-lipidated plasma was acquired from SeraCare Life Sciences . Packed red blood cells (RBC) from Three different sets of quality control materials for the four steroids at varying concentrations were prepared. Whole blood was enriched with 15 ng/mL or 150 ng/mL of 17-OHP, 11, 21, and 4-A to prepare the low- and high-quality control materials respectively. The normal control was not inoculated with 17-OHP, 21, or 4-A. Cortisol was inversely spiked into the normal, low- and high-quality control materials at concentrations of 80, 15 and 5 ng/mL, respectively, to mimic patient profiles with high 17-OHP and low cortisol concentrations.Calibrators were prepared with equimolar enrichment of each steroid in the following levels: 0, 2.5, 5.0, 10, 50, 100 and 250 ng/mL. These calibrators were also used to assess the method’s linearity and limit of quantification.The unenriched and enriched whole blood pools used in creation of the quality control materials and calibrators were dispensed onto filter paper, dried overnight under ambient conditions, and stored at –20 °C in zip-closure plastic bags containing desiccant packets.As part of routine screening within Wisconsin, whole blood samples obtained from newborns between 0 and 168 h after birth were collected on Whatman GmbH grade 903 filter paper, dried, and sent to the Wisconsin NBS laboratory. 17-OHP was measured on all specimens using a time-resolved fluoroimmunoassay according to the manufacturer’s specifications . The results of this first-tier CAH screening were interpreted according to the birth weight of the newborn . Any speA 3.2 mm (⅛”) punch of the DBS specimen was placed into a 96-well, round-bottom polypropylene plate. The steroids  were extracted from the punch using 175 µL of an 80:20 acetonitrile/water solution containing 3 ng/mL of each internal standard, followed by shaking at 50 °C for 60 min. The extraction solution was transferred to a clean 96-well plate and dried under nitrogen gas for 35–40 min at a temperature of 40 °C. The dried well was reconstituted using 50 µL of 40:60 methanol/water solution with 0.3% formic acid and then shaken for 10 min at room temperature, prior to analysis.The steroids were separated by liquid chromatography using a Phenomenex Kinetex  5 µm C18 110A 50 × 2.1 mm column and analyzed using an AB Sciex API 4500 tandem mass spectrometer with a TurboV electrospray ionization source. The API 4500 was operated in positive-ionization mode with the ion spray voltage set to 1500 V, declustering potential 70 V, excitation potential of 10 V, and a collision cell exit potential of 13 V. Additional steroid dependent parameters are in Adequate baseline separation of the four steroids, the column wash and re-equilibration was achieved with a total run time of nine minutes. The injected sample volume was 20 µL. The steroids and associated internal standard were analyzed using the multiple reaction monitoring (MRM) pairs in The analytical method described above extracts, identifies, and quantifies four steroids. The described assay validation results below highlight these four steroids . To identify newborns with congenital adrenal hyperplasia due to 21-OH deficiency, the following three steroids  and two ratios [(17-OHP + 21)/F and (17-OHP + 4A)/F] were used for interpretation. p < 0.0001 and all tests are two-sided. For the population assessment, the four steroid values were log transformed to improve symmetry of the data and stabilize variance across the range of birth weight. Statistical analysis of the data was performed using non-parametric analysis of variance  and Mann–Whitney tests for follow-up pairwise comparisons. Significance was defined as R2) of >0.9970. The recovery of each steroid was within ±15% of expected concentration across the linear range.A complete clinical assay laboratory validation was performed for four steroids quantified within the second-tier assay . Assay precision was assessed using the two quality control specimens and the unenriched specimen analyzed in six replicates on seven different days. The intra- and inter-assay precision was less than 11% and 15% respectively for all four steroids at varying concentrations exceeding the limit of detection . The limit of quantification was assessed at 2.5 ng/mL for each steroid, with a CV of 26% or less. Linearity ranged from 0 to 500 ng/mL with slopes averaging between 0.83 and 1.15 and a coefficient of determination  after birth. The birth weights for these patient specimens ranged from 300 to 4810 g: 27% with a birth weight less than or equal to 1500 g; 28% with a birth weight between 1501 and 2500 g; and 45% with birth weight >2500 g. The 17-OHP values as measured by the first-tier immunoassay were within the normal range as determined by the birth weight adjusted cutoff  as assessed by the one-way ANOVA Kruskal–Wallis test. Androstenedione also declined in concentration with increasing birth weight and was statistically different between each of the three birth weight groups . Cortisol and 21-deoxycortisol were not statistically related to birth weight .For each steroid , the data was plotted and a linear regression of the log transformed value against a 5-knot restricted cubic spline for birth weight was fit, with the model stratified according to age at sample collection (before 24 h versus 24–168 h) . 17-hydrp-value <0.0001) as assessed by the Mann–Whitney test. Further stratification by timing of collection and birth weight, using the weight categories listed above, showed statistical differences between groupings for 17-OHP and 4-A . The Mann–Whitney tests performed on cortisol and 21-deoxycortisol were not consistently significant among the groups (stratified by birth weight and collection time).Steroid concentrations were also assessed by the timing of collection, either before 24 h, or at 24 h or greater; 61% of specimens were collected at 24 h or greater; and 39% of specimens were collected at less than 24 h. The 17-OHP, 4-A, F, and 21 concentrations in specimens collected before 24 h as compared to 24 h or greater were all statistically different . Within the 144 specimens, 9 (6.3%) were collected prior to 24 h and 135 (93.7%) were collected at 24 h or later. The birth weight in this population ranged from 403 to 4060 g; 19.4% had a birth weight ≤1500 g, 13.9% were between 1501 and 2500 g, and 66.7% were greater than 2500 g. None of the false positive specimens had all five markers (three steroids and two ratios) greater than 3stdev above the mean, nor were any of the individual markers greater than 10 standard deviations. However, 7 out of 144 specimens (~5%) had abnormal results, greater than 3stdev, for four of the five markers. The remaining 137 specimens had three or fewer markers above the 3stdev cutoff.An interpretation algorithm was proposed after analysis of the 18 specimens from confirmed cases, 144 false positive specimens, and 783 presumptively normal specimens. All newborns will continue to receive the routine first-tier immune assay to measure the 17-OHP concentration. If the 17-OHP value is determined to be abnormal, depending upon the birth weight adjusted cutoff, the specimen will be referred for second-tier testing. The second-tier test will measure four different steroid concentrations and two ratios. Specimens with 17-OHP, 4-A, 21, and the two ratios [(17-OHP + 4-A)/F and (17-OHP + 21)/F] above three standard deviations will be reported as abnormal (screened positive). Patients will be immediately referred for endocrine consultation and confirmatory testing. Likewise, any sample with any of the five markers (three steroids or two ratios) above 10 standard deviations will also be referred for immediate endocrine consultation and confirmatory testing. To allow for a conservative interpretation and to limit the possibility of false negative results, specimens with four of the five markers above the three standard deviation cutoffs would result in a request for repeat newborn screen See the algorithm in As per the Wisconsin protocol for babies in special care units, a routine repeat screen at 14 days of life will continue to be performed on all newborns with a birth weight <2200 g, regardless of the initial CAH result. These repeat specimens will be tested and evaluated using the same algorithm, with the exception of the first-tier 17-OHP cutoff. The repeat specimens will have a more stringent first-tier 17-OHP cutoff, dropping from initial birth weight cutoff to the next lower cutoff .A one-year prospective study incorporating second-tier testing for CAH was implemented into routine newborn screening using the algorithm in A calculation of the false positive rates within the prospective study  was determined to be 0.75% following first-tier testing, decreasing to 0.04% after second-tier testing and resulting in a greater than 95% reduction in false positive cases. The calculated positive predictive value [true positives/] before and after second-tier testing was also dramatically improved, from 1% to 17%.The introduction of newborn screening for CAH has profoundly impacted the well-being of affected individuals (approximately 3 to 6 newborns each year in Wisconsin) by reducing the morbidity and the risk of neonatal death due to salt-wasting crisis. However, the current first-tier screening protocol has a high false-positive rate (approximately 0.75% in Wisconsin), leading to unnecessary testing and clinical evaluations, along with parental anxiety and stress ,25. The The success of the second-tier test for CAH has been tempered recently with a concern for false-negative results. Previous reports of missed cases have largely been limited to the milder forms of CAH missed by first-tier analysis ,29,30. HThis study describes the Wisconsin experience with the validation and implementation of the second-tier LC–MSMS test, including the development of an algorithm for the interpretation of results. Wisconsin chose to analyze four different steroids . Assay performance metrics, including precision, accuracy, and linearity, were all consistent with previously reported studies. As part of the assay validation, a complete analysis of the steroid concentrations within the normal population was performed. Consistent with literature reports, steroid concentrations change over time and are also impacted by physiological differences between a premature, low-birth-weight baby compared to a full-term baby . TherefoFrom the assessment of the normal population, along with false positive and confirmed cases, an interpretation algorithm including multiple steroids  and two additional ratios, stratified by birth weight and specimen collection timing, was proposed. Sixteen of the confirmed cases of classical CAH (SV or SW) had all five markers elevated above 3 standard deviations from the mean. There was, however, a dramatic range in steroid concentration within the confirmed cases (17-OHP ranged from 6.14 to 395.00 ng/mL). The Wisconsin screening algorithm also incorporates provisions for when a single marker is elevated at a concentration greater than 10 standard deviations from the mean. One patient confirmed to have SV CAH was collected at 2 h of life. In this specimen, 3 markers were elevated, but both ratios were greater than 10 standard deviations from the mean. In addition, the Wisconsin modified algorithm, incorporating a 10 standard deviations cutoff from the mean, also detected a non-classical CAH patient. To address the potential for false negatives due to missed interpretations, the algorithm includes a “possible abnormal” reporting option to be used when four of the five markers are outside three standard deviations. The one-year prospective study achieved a 95% reduction in false positive rate to 0.04%, even with a conservative algorithm allowing for a “possible” abnormal interpretation. This tempered approach may be an appropriate alternative to minimize false negatives, but not substantially increase the false positive results. During the prospective study, 5 CAH patients were identified out of 63,725 analyzed . This is higher than the reported incidence of 1 in 20,000, and suggests that it is likely no cases of CAH were missed. The impact of the second-tier test on the entire newborn screening program is significant. Studies have demonstrated that the second-tier test for CAH reduces the burden of follow-up care and has improved clinical practice by minimizing the need for sub-specialty, endocrine evaluations . In addiThe risk of missed CAH cases, whether after first- or second-tier analysis, will always remain a concern for programs. The decrease in flux through the steroid synthesis pathways, largely due to cortisol inhibition or the lack of mature enzyme function, will continue to contribute to false negative rates. The incorporation of a more complete steroid panel, including measuring steroids within the mineralocorticoid pathway, may allow for a more thorough assessment of the pathway. Additionally, clinically relevant factors—such as birth weight or the timing of collection—should be incorporated into all screening algorithms, similar to what has been proposed by Rinaldo et al. . In the This study replicates and expands upon previous work to validate and implement a second-tier assay for classical CAH. Multiple steroids and ratios, along with appropriate adjustment for physiological factors, collection time and birth weight, enabled Wisconsin to suggest a more comprehensive screening algorithm that has not been previously delineated.


S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine—which also targets 2C—but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike ( There is a great need for antiviral drugs to treat enterovirus and rhinovirus infections, which can be severe and occasionally life-threatening. This study describes novel small molecule inhibitors that target a broad spectrum of clinically relevant enterovirus species; a common mechanism of resistance development revealed the target to be a highly conserved binding pocket in the viral helicase 2C. Enterovirus of the family Picornaviridae is a large group of nonenveloped, positive-sense, single-stranded (+) RNA viruses. Four enterovirus species (EV-A to -D) and three rhinovirus species (RV-A to -C) constitute the set of human pathogens that have large medical and socioeconomical impact, such as poliovirus (PV), coxsackievirus (CV) A and B, echoviruses, numbered EVs  and RVs  . Ex. Ex42]. t ≥ 1 μM  and overEVs, especially EV-A71, EV-D68, and CV-A24v, impose serious public health threats. Currently, there are no antiviral therapies licensed to treat EV infections. The highly conserved nonstructural protein 2C fulfills pleiotropic functions during the viral life cycle. Hence, it represents an interesting target for the development of broad-spectrum anti-EV inhibitors. The FDA-approved drug fluoxetine, which is an SSRI, was an attractive candidate for the treatment of EV infections, but several recent studies raised concerns about its clinical application. One retrospective study revealed adverse effects when using fluoxetine as treatment option for EV-D68-associated paralysis. The data suggested a worsening of patient condition, possibly due to its SSRI activity . AdditioS)-fluoxetine, are not neuroactive.In a high-throughput screen, compound 1 was identified as a CV-B3 inhibitor . The cheS)-fluoxetine resulted in a broader antiviral spectrum than the less active racemic mixture lends support to this hypothesis ), Rhabdomyosarcoma (RD) cells (American Type Culture Collection [ATCC]) and HeLa R19 cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% (vol/vol) fetal calf serum . HAP1 cells were obtained from Horizon Discovery Group plc  and cultured in Iscove’s Modified Dulbecco’s Medium  containing 10% (vol/vol) FCS. Huh7-Lunet 7/T7, a stable cell pool expressing T7 RNA polymerase and blasticidin S-deaminase , were kieviously , 48. BriS)-fluoxetine were purchased from Sigma-Aldrich. GuaHCl was dissolved in water at 2 M stock concentration, and all other compounds were dissolved in DMSO at 10 mM stock concentration. Neurobasal-A medium, penicillin–streptomycin , B27 plus supplement, N2 supplement, and l-glutamine are obtained from Life Technologies . Polyethyleneimine (PEI) solution (50%), sodium borate, boric acid, and all other chemicals (unless stated otherwise) are obtained from Sigma-Aldrich . General chemical synthesize route and NMR spectra of the compounds are listed in GuaHCl and (EV-A71 (strain BrCr), PV-1 , and the EV-D68 strains Fermon, 431100074, 4310901348, 4310902042, 4310900947, and 4310902284 were obtained from the National Institute for Public Health and Environment (RIVM) in the Netherlands. HRV-2 and HRV-14 were obtained from Joachim Seipelt from the Medical University of Vienna in Austria. CV-B3 (strain Nancy) was obtained by transfecting BGM cells with RNA transcripts derived from the full-length infectious clones p53CB3/T7 as described by Wessels and colleagues . EV-A71 2 flask in the presence of 19d. In parallel, virus without compound was taken along and used as a reference. The virus titers from the obtained virus variants were determined by endpoint dilution, and the P2 and P3 regions were sequenced. The resistance and cross-resistance phenotype of the virus variants was determined with a multicycle viral replication assay.We raised compound 13–resistant EV-A71, CV-B3, and EV-D68 viruses as described earlier . In shorThe CV-B3 mutations 2C[C179F], 2C[C179Y], 2C[F190L], 2C[I227V], and 2C[A224V/I227V/A229V] were previously introduced into the p53CB3/T7 infectious clone . Mutatio50) [The EV-A71 mutations 2C[T133S], 2CR151H], 2C[M193L], 2C[M193L/R151H], and 2C[M193L/R151H/T133S] and the EV-D68 mutations 2C[V96M] and 2C[T196S] were introduced into the pEV-A71 backbone (kindly provided by Johan Neyts) [H, 2CM19, respect50) .50) [Renilla luciferase Assay System  was used to determine the luciferase activity. Cell viability was determined in parallel using the AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol. Optical density at 490 nm was determined using a microplate reader.Virus infections were performed by incubating subconfluent HeLa R19 cells with virus at the indicated MOI at 37 °C for 30 minutes. Next, the inoculum was removed, and fresh control medium or compound-containing medium was added to the cells. At the indicated time points, cells were frozen. For measurements of infectious particles, virus was released from the cells by three freeze-thawing cycles. Virus titers were determined by endpoint dilution assay and calculated by the method of Reed and Muench and expressed as 50% tissue culture infectious dose (TCID50) . In the Renilla luciferase by 50% (EC50) was calculated by nonlinear regression analysis with GraphPad Prism Version 6. We set the threshold for compound activity at concentration of 10 μM or less since low μM range is typical for antivirals in clinical use. Cytotoxicity of the compounds was assessed in a similar set-up, and CC50 values were derived from cell viability values determined with an MTS assay.Subconfluent layers of HeLa R19 cells were seeded in 96 wells and treated with serial dilutions of the corresponding compounds. Cells were infected with viruses at the lowest possible MOI resulting in full CPE within 3 days . Subsequently the cells were incubated at 37 °C for 3 days until full CPE was observed in the virus-infected untreated cell controls. Cell viability was determined using the AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol. The optical density at 490 nm was determined using a microplate reader. Raw OD values were converted to percentage of untreated and uninfected cell control after subtraction of the background. The concentration of compound that inhibits virus-induced cell death or expression of Escherichia coli RosettaTM 2 (DE3) . When cultures reached OD600nm of 0.5, protein expression was induced by the addition of 0.5 mM IPTG. Subsequently, protein was expressed for 16 h at 18 °C, with shaking at 200 rpm. Further protein purification steps were performed essentially as described previously [The DNA fragment encoding residues 116 to 329 of CV-B3 2C (strain Nancy) was cloned into a pET28b plasmid, downstream of an N-terminal, and 3C protease cleavable, hexahistidine-MBP tag. The F190L mutation was introduced using a Q5 site-directed mutagenesis kit . The recombinant WT protein, and F190L variant, were produced in eviously , with theviously . Each we2 incubator. At DIV4, 90% of the serum-free dissection medium was replaced with serum-free culture medium . Rat primary cortical neurons were used for neurotoxicity assessment at DIV15.Primary rat cortical cells were isolated from PND0-1 Wistar rat pups as described previously , 55. BriEach well of a 48-well MEA plate contains 16 nanotextured gold micro-electrodes  with 4 integrated ground electrodes, yielding a total of 768 channels for simultaneous recording (for review see ). SpontaPrior to the recording, MEA plates were allowed to equilibrate for 5–10 minutes in the Maestro. Following a 30-minute baseline recording, wells were exposed to the test compounds or the DMSO control, and activity was recorded for another 30 minutes to determine the acute effects of test compounds on neuronal activity. Stock solutions of compounds dissolved in DMSO (10 mM) were diluted (1:100) in serum-free culture medium to obtain 100 μM solutions, which were used for exposure of cells in the MEA . In order to prevent receptor (de)sensitization, each well was exposed only to a single concentration.exposure/parameterbaseline) × 100%. Hereafter, TRs will be normalized to DMSO control. Wells that show effects 2× SD above or below average were considered outliers (5.1%) and were removed for further data analysis.To determine (modulation of) spontaneous activity, RAW data files were re-recorded to obtain Alpha Map files for further data analysis. In this re-recording, spikes were detected using the AxIS spike detector  with a variable threshold spike detector set at 7× SD of internal noise level (rms) on each electrode. Post/pre-spike duration was set to 3.6/2.4 ms. For further data analysis, spike files were loaded in NeuralMetric Tool . Only active electrodes (MSR ≥ 6 spikes/min) in active wells (≥1 active electrode) were included in the data analysis. The (network) bursting behavior was analyzed using the Poisson Surprise method  with a mUptake activity of hNET, hDAT, and hSERT was measured using the Neurotransmitter Transporter Uptake Assay Kit from MDS Analytical Technologies  as described previously , 48. Bri2/95% air atmosphere at 37 °C. Experiments were performed the next day (day 1). Cells were pre-incubated with the fluorescent substrate for 12 minutes (t = −12 to t = 0) prior to a 30-minute drug exposure. Culture medium was replaced by 100 μL/well fluorescent substrate solution, and uptake measurements were started. At t = 0, 100 μL/well HBSS with 0.1% DMSO (control) or with test compound  was added to each well, and uptake was measured continuously for 30 minutes. Background wells were pre-incubated with 100 μL/well HBSS without fluorescent substrate solution and exposed at t = 0 min to 100 μL/well HBSS.On day 0, HEK 293 cells were seeded at a density of approximately 60,000 cells/well in clear-bottom, black-walled, 96-well plates  coated with PLL buffer (50 mg/L). Cells were allowed to proliferate overnight in a humidified 5% COFluorescence was recorded every 3 minutes, starting directly after addition of the fluorescent substrate solution (t = −12) using a microplate reader  at 37 °C at 430/515 nm excitation/emission wavelength in bottom-reading mode using optimal gain values for each cell type . Cell attachment was visually examined following experiments.The fluorescence of each well was background corrected (time- and plate-matched). First, uptake of the fluorescent substrate was first determined per well by calculating the change in fluorescence (ΔFU) at 12 minutes after exposure to the test compound (t = 12) compared to the fluorescence prior to exposure , as a percentage of the fluorescence prior to exposure. Second, the percentage uptake in control wells of all plates was averaged, and wells that showed values 2× SD above or below average were considered outliers and were excluded from further analysis. Uptake in compound-exposed wells was expressed as a percentage of control wells, outliers in exposed groups (effects 2× SD above or below average) were removed (approximately 1%), and all uptake values were scaled between 0% and 100%.For prediction of solvent accessible tunnels, we used the crystal structure of EV-A71 2C PDB: 5GRB/Chain B and PV 2C PDB 5Z3Q/Chain A. As a starting point for the tunnel prediction, the amino acid of 2C of EV-A71 M193 and PV-1 2C F164—N179 and M187, respectively—were used. We used the MOLEonline web interface , CAVER 3P ≤ 0.05 was considered significant. Statistical analysis, nonlinear regression, and the graphs were made with GraphPad Prism Version 6 or R version 3.6.0 (R core team 2019).Each experiment was performed at least in technical duplicates, and all experiments were performed in biological triplicates. Statistical significance of the MEA data and transporter inhibition was determined using one-way ANOVA and, if applicable, a post hoc Tukey test. S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Table50 and CC50 values in μM. Data represent mean ± SD calculated from two different experiments both performed in biological triplicates. All underlying experimental data are displayed in Multicycle viral replication assays were performed in HeLa R19 cells, and shown are EC(DOCX)Click here for additional data file.S2 Table50 and CC50 values in μM. Data represents mean ± SD calculated from two different experiments both performed in biological triplicates. All underlying experimental data are displayed in Multicycle viral replication assays were performed in HeLa R19 cells, and shown are EC(DOCX)Click here for additional data file.S3 Table50 and CC50 values in μM. Data represent mean ± SD calculated from two independent experiments both performed in biological triplicates. SI = selectivity index (CC50/EC50). All underlying experimental data are displayed in Multicycle viral replication assay was performed in HeLa R19, HAP1, and BGM cells. Shown are EC(XLSX)Click here for additional data file.S4 TablePrimers used for introducing mutations in CV-B3, EV-A71, and EV-D68. Mutations introduced are shown in small characters in the primer sequence.(XLSX)Click here for additional data file.S1 Fig3BH, MeOH, rt, on, 30%; (iii) 2-bromoacetyl chloride, TEA, DCM, rt, 1h, 68%; (iv) methylamine, EtOH, rt, on, 66%; Synthetic route b): (i) K2CO3, benzyl bromide, DMF, rt, 18h 68%; (ii) Boc, DMAP, TEA, THF, rt, 18h, 90%; (iii) Pd/C 10%, H2 atmosphere, EtOAc/MeOH  compound 4 or compound 18b, TBTU, DIPEA, DMF, 45 °C, 48–72h, 79%–45%; (v) DCM/TFA (1:1), rt, 4h, 91%–89%. Synthetic route c): (i) NaBH4, MeOH/THF (4:1), rt, 6 h, 88%; (ii) 4-fluorobenzoyl chloride, TEA, DCM, rt, 3h, quantitative.Synthetic route a): (i) NaBH4, MeOH/THF (4:1), rt, 6 h, quantitative; (ii) Compounds 5a–5d: corrisponding acyl chloride or sulphonyl chloride, TEA, DCM, rt, 3h, 63%–98%; Compounds 1, 5e–f: corrisponding carboxylic acids, TBTU, DIPEA, DMF, rt, on, 37%–69%; Compounds 5g: Na(AcO)(TIF)Click here for additional data file.S2 Fig(i) NaBH4, MeOH/THF (4:1), rt, 6 h, 40%–99%; (ii) furan-2-carbonyl chloride, TEA, DCM, rt, 3 h, Y = 56%–99%; (iii) thionyl chloride, DCM, reflux, 2h; (iv) compound 18d, TEA, DCM, rt, 3h, 75%.(TIF)Click here for additional data file.S3 FigMultiple sequence alignment of EV-A71 (strain BrCr), CV-B3 (strain Nancy), PV (strain Sabin), and EV-D68 (strain Fermon) was performed with Clustal Omega. Invariant amino acids are highlighted in red. Secondary structural elements are shown on top of the alignment and are based on the EV-A71 crystal structure (PDB: 5GRB). Functional motifs are indicated in black. The green box indicates resistance mutations which are located at or close by the α2 helix. The yellow boxes highlight distal mutations.(TIF)Click here for additional data file.S4 FigThe mole online version (40) was used to calculate solvent exposed tunnels in 2C of PV-1 (PDB: 5z3Q) from different starting points. (A) The starting point for calculation was the amino acid F164, and in (B) the starting point for the tunnel prediction was N179. The Pymol plug-in CAVER 3.0.3 was used to calculate solvent accessible tunnel in the nonstructural protein 2C (58). (A) The EV-A71 2C crystal structure PDB: 5GRB, chain B was used. The amino acid M193 in the α2 helix of 2C is depicted in green and represents the starting point to identify solvent accessible tunnels. The identified tunnels are shown red, blue, green, and yellow. (B) For PV, the 2C crystal structure 5Z3Q, chain B was used to identify solvent accessible tunnels using the amino acid M187 as starting point. The identified tunnels are depicted in red, green, and blue. The CASTp online tool was used to predict cavities on the protein surfaces of the 2C nonstructural proteins of (C) EV-A71 and (D) PV (59). The cavities are highlighted in blue.(TIF)Click here for additional data file.S5 Fig(A) Schematic overview of the microelectrode array (MEA) recordings used to measure changes in neuronal activity. Photographs of the Maestro 768-channel amplifier (A) and 48-well MEA plate (B). Each well contains 16 nanotextured gold micro-electrodes on top of which neuronal cells can be cultures for recording of spontaneous electrical activity (C). Baseline activity recorded before exposure  is compared to activity following exposure to a (inhibitory) test compound  to determine a TR that describes the changes in neuronal activity due to exposure to the test compounds. Modified after Tukker and colleagues, 2016 . TR, tre(TIF)Click here for additional data file.S6 Fign = 12–16 wells, derived from 2 independent cultures, *P < 0.05). (B) Modulation of spontaneous neuronal activity by fluoxetine and antiviral compounds. When tested at a single, high concentration (10 μM), fluoxetine induced a profound inhibition of MSR (left), MBR (middle), and MNBR (right), whereas the antiviral compounds 12a, 12b, 19b, and 19d were without effect. Neuronal activity is depicted as the mean TR ± SEM as percent of control (DMSO) wells . All underlying experimental data that are displayed can be found in (A) Modulation of spontaneous neuronal activity by fluoxetine. Concentration–response curves for inhibition of MSR (left), MBR (middle), and MNBR (right) following acute exposure to fluoxetine. Neuronal activity is depicted as the mean TR ± SEM as percent of control (DMSO) wells ((TIF)Click here for additional data file. 22 May 2020Dear Dr van Kuppeveld, Thank you for submitting your manuscript entitled "Rational design of highly potent pan-enterovirus inhibitors targeting the non-structural protein 2C" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, i.e. by May 24 2020 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Di Jiang, PhDAssociate EditorPLOS Biology 24 Jun 2020Dear Dr van Kuppeveld,Thank you for sending us the new information in your appeal to our previous decision on your manuscript "Rational design of highly potent pan-enterovirus inhibitors targeting the non-structural protein 2C" which was evaluated by the PLOS Biology editors, an Academic Editor, and by three independent reviewers.As you know that we have rescinded our last decision, and we now welcome re-submission of a much-revised version that takes into account the reviewers' comments AND includes the new compound with significantly improved potency compared with that in the version of the manuscript reviewed by our external reviewers. We will need to see that our reviewers are satisfied with the potency of the new derivative, and you will need to address all other concerns raised by the reviewers except the two mechanistic aspects that we overruled on and the exploration of SSRI activity which we overruled in our last decision. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.We expect to receive your revised manuscript within 2 months. plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.Please email us  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*PLOS Data Policy*http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. For instructions see: Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Di Jiang, PhDSenior EditorPLOS Biology*****************************************************REVIEWS:Reviewer #1: Picornaviruses are significant human pathogens for which there is no vaccine or antivirals. This manuscript described the lead optimization based on compound 1, which was identified through a HTS. Several analogs including 12, 19b and 19d, were identified to have broad-spectrum antiviral activity against EV-A71, EV-D68, CVA24, Polio and HRV. The mechanism of action was mainly studied by the resistance-selection experiment. Additional evidence was provided by the thermal shift binding assay. The highlights of this study include:1) Broad-spectrum antiviral profiling against multiple enteroviruses. 2) Detailed pharmacological characterization. Using compound 19d as a chemical probe, resistant mutants have been selected in CV-B3, EV-A71 and EV-D68 viruses. Mutants were identified in the 2C protein, and the corresponding mutant viruses were generated through reverse genetics. Although the leads generated from this study are promising drug candidates for picornavirus antiviral, there are several issues that remain to be addressed:1) The antiviral activity of the optimized hits 12,19b and 19d are on par similar to the (S)-fluoxetine (Table 5), although it is noted that the antiviral spectrum is expanded. The potency needs to be further optimized down to sub-micromolar to have clinical relevance. There are several series of 2C inhibitors in the literature that have submicromolar efficacy. Therefore, the results in this study are not particularly significant. 2) The detailed mechanism of action at the molecular level remains elusive. Although resistant mutants have been identified, no docking models were shown to illustrate how compound 19d interact with the 2C protein. The results shown in Fig. 6 have no direct relationship with the drug binding. Overall the detailed mechanism of action remains a mystery and this study does not significantly advance our knowledge on the pharmacology of 2C inhibitors. 3) The authors did not explain from the structural perspective why compounds 12, 19b and 19d have a broader spectrum of antiviral activity compared to fluoxetine. 4) The overall approach is not particularly novel, it represents a standard medicinal optimization approach. Therefore this manuscript is more suitable for chemistry journals. Specific comments are:1) It was claimed that one of the advantages of current hits is that they are not neuroactive. I am not sure what this actually means. Does this have to do with the potential side effects of fluoxetine analogs? The compounds described here are still at the early stage of drug development, and there are many parameters need to be optimized including microsomal stability, solubility, permeability, CYP inhibition and etc. Therefore neurotoxicity side effect is not a major concern at this stage. 2) Line 113, "CPE-reduction assays" should read "CPE assay". There is only plaque reduction assay, not CPE reduction assay. This should be changed throughout the manuscript.3) In table 2, the structures for R1 substitution are missing.4) In Fig. 7, why the other two mutants H243R, F190L+H243R have not been tested? Reviewer #2: The authors report the identification of antiviral compounds with pan-enterovirus and pan-rhinovirus activity starting from a previously reported compound 1. The escape mutant analysis has permitted the authors to suggest that enteroviruses and rhinoviruses could share a druggable pocket in the 2C protein that needs further validation.Major comments:Overall this is a difficult paper to read and fully appreciate the key finding proposed by the authors that there is a common druggable pocket in entero- and rhinoviruses that can be targeted to try and obtain potent drugs. An alignment of 2C protein from the various strains examined in this study in relation to previous mutations in CV-B3 virus and the strains compared in this study may be useful for non-EV readers to try and integrate the findings in the different viruses. Data on SSRI activity of the new compounds could add value to attractiveness of this compound which presumably needs to be further optimized for potency.Minor comments:Table 2 is incomplete.Line 42 : …numbered enteroviruses and rhinoviruses? Line 335: … should it be "mutants were not tested"Line 714: … should it be "end-point dilution assay"Reviewer #3: Enteroviruses are ubiquitous pathogens. Despite decades of trying, no direct acting antiviral medications exist. This is an extremely well written paper that describes the exploration of anti-enteroviral activities and cytotoxicity of derivatives of a compound described originally by Zuo et al (ref 20). The authors, noting the structural similarities between this compound and fluoxetine  carefully modified key elements of this molecule and in the process identified congeners with broader antiviral activity, reduced impact on neuronal activity and lower cytotoxicity. The paper closes somewhat speculatively by analyzing models of the target of action of these compounds, the viral 2C protein. The paper is highly detailed and may have initially been composed with the goal of publishing it in a medicinal chemistry journal. The detailed description of the schema for synthesis does not detract from the paper, and instead makes it all the more impressive. The only criticisms of this detail may be the complexity of the figure legends, and several small errors in labeling. For example, in line 163 the text refers to "final product 9", which appears to refer to compound 15 . Also, in two places "13d" (e.g. line 347) is mentioned, when 19d is intended. Several other minor errors are present in Table legends for Table 4 and 5 with begin wiht "Multicycle (sic) was performed in Hela R19 cells,.." (Multicycle replication assay were performed..." may have been intended).Also, a number of terms and abbreviation may need definition for the audience of this journal.Two somewhat important points that should be addressed follow:First, the authors should define levels of antiviral activity. Most experts would not consider EC50 values of 5 to 10 micromolar to be more than mild to moderate; submicromolar is more typical of molecules that come to be used therapeutically. The paper would be improved by mentioning this or similar stratification.One related point: the EC-50 values for compound 12 CV-B3 (Nancy) of ~10 to 20 nM are very impressive, but since this strain no longer circulates, one wonders why other enterovirus B virus strains were not examined, as was done with EV-A71, EV-D68, and CVA24. Addressing these two points would not detract from this excellent paper, but as it is, the authors seem a bit overly enthusiastic in their characterization of their best compounds as candidates for clinical development. 8 Jul 2020Attachment20200707_ResponsetoReviewers.docxSubmitted filename: Click here for additional data file. 19 Aug 2020Dear Frank,Thank you very much for submitting your revised manuscript "Rational design of highly potent pan-enterovirus inhibitors targeting the non-structural protein 2C" for consideration as a Research Article by PLOS Biology and please accept my apologies for the time it has taken us to return to you with a decision on the work. Your work has now been seen by reviewers 1 and 3, whose comments you will find at the end of this email, an academic editor and the staff editorial team.Based on the reviews, I am delighted to say that we can in principle offer to publish the work, conditional on addressing a few outstanding referee and editorial (reporting and formatting) requests. Please address the issues raised by reviewer 3, which are self-explanatory. Before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. In addition while checking through the manuscript and associated files, we noticed the following editorial points which we will need you to address. As we can't proceed until all these requirements are met, your careful attention to addressing all issues (the below and the ones sent by my colleague in a subsequent email) will help prevent delays to publication. 1. As indicated by reviewer 1 (opinion shared by us and our Academic Editor), the manuscript seems currently geared to an organic chemistry/chemical biology audience. As our readership is eminently composed of biologists, the manuscript will need to be thoroughly reworked with this in mind (without removing data). As part of these efforts, and in an attempt to make the work more accessible for a life science readership, we would strongly suggest to move Schemes 1 and 2 (which should be relabelled as two panels within a figure) and tables 2, 3 into the supplementary information.2. We would suggest a title that more accurately reflects the manuscript, such as "Rational design of highly-potent broad-spectrum enterovirus inhibitors targeting the non-structural protein 2C", in addition to addressing reviewer 3s point about the pan-inhibition claimed in the manuscript text.3. Please note that there are currently two Table 1 (on in the Methods), and relabelling will be needed, accordingly. The Methods table could also be included in the supplement.4. Please include an "Ethics Statement", labelled as such, as a first subheading in the Methods section. This should also include the protocol number whereby your Ethical Committee granted approval of these experiments (in addition to the other relevant informaiton you currently have elsewhere in the Methods). 5. The "Calculations and Graphs" section should be relabelled "Statistical Analyses" and include all of the statistical analysis information that is currently scattered in other subsections in the Methods. The information in the figure legends should not change .6. Please nclude a "Cells" subsection detailing the provenance  of every cell line used, whether they have been authenticated in your laboratory  and whether they have been tested for mycoplasma contamination.7. We will need a written statement of whether the photos in SF3 have been generated by you or have been published elsewhere. If so, we can only use them if they have been published under a CC-BY license.http://journals.plos.org/plosbiology/s/data-availability.8. You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: All individual quantitative observations that underlie the data summarized in the figures and results of your paper need to be made available. Thus, please provide with your final manuscript supplementary files , uploaded as 'Supporting Information' and referred to  using the following format: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore). The Excel files must be clearly labelled such that the experiments and data points they refer to are easy to understand. Your manuscript needs underlying data for the following figures: Fig 2, Fig 3, Fig 4 B, C, E, F, H, I, Fig 6, 7B, 8B, SF4. Each of those figure legends needs to state where the underlying data can be found .We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by reviewer 3 and the editorial points above. *Submitting Your Revision*https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include:To submit your revision, please go to - a cover letter detailing the changes made in response to the editorial requests made in this letter- a Response to Reviewers file that provides a detailed response to any reviewers' comments, if applicable - a track-changes file indicating any changes that you have made to the manuscript. ADDITIONAL INFORMATION*Copyediting*Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:https://journals.plos.org/plosbiology/s/supporting-information*Published Peer Review History*Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*Early Version*Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.*Protocols deposition*https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methodsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Please do not hesitate to contact me should you have any questions.With best wishes,NoniaNonia Pariente, PhD,Senior Editor,djiang@plos.org,PLOS Biology------------------------------------------------------------------------Reviewer remarks:Reviewer #1: During the revision, the authors made an effort to develop an additional compound 12b that improved antiviral activity. The highlights of this manuscript include:1) A lead compound 12b with broad-spectrum antiviral activity against enteroviruses and rhinoviruses were identified. 2) The mechanism of action was studies by resistance selection using CVB3, EV-A71, EV-D68 viruses. Drug resistance was confirmed by reverse genetics and thermal shift binding assay.3) Mutations were mapped to the alpha2 helix and solvent accessible tunnels were identified in this region.Weaknesses of this manuscript include:1) Compared to the previously publications related to 2C inhibitors, this current study did not significantly advance our knowledge how 2C inhibitors interact with the drug target. The mutations for the current compounds were mapped to the same region as previously reported 2C inhibitors.2) The effects of the mutations on the stability and function of 2C protein were not examined. Overall this manuscript is more suitable for a chemistry journal like the Journal of Medicinal Chemistry or ACS Pharmacology and Translational Research. Reviewer #3: The authors of this manuscript now submit a revised version that corrects the typographic errors in the first version, clarifies a number of points, and most importantly, demonstrates the antiviral properties of a new compound (12 b) resulting from the logical incorporation of properties of two derivatives of a molecule identified by another group (ref 20) via high-throughput screening. This new compound exhibits appreciable activity against representative strains from enterovirus species A, B, C, and D, and two of the 3 rhinovirus species. The paper is much improved by these changes. However, several straightforward changes should be made, in this reviewers opinion.1. The authors state in their responses that "we agree that mentioning the therapeutically relevant range  would improve the paper. We defined the therapeutically relevant window in line 793-796". However, lines 793 to 796 are in the section labeled "MEA recordings of spontaneous neuronal activity in rat primary cortical cultures". What is needed is a straight forward statement in the methods, such as saying that antiviral activity is defined as 50% inhibition of virus production or expression of luciferase at concentrations of 10 micromol/ L or less. Clearly submicromolar activity is typical of most antivirals in use, and it is exciting to see examples of low nanomolar range reported for compound 12b..2. In the introduction, the authors state "Even though fluoxetine was well tolerated, it revealed no clinical benefit. Rather, the data pointed to a worsening of the patient conditions in the fluoxetine-treated cohort.(26) This is paradoxical; if people became more ill with fluoxetine, how can we say that it was well tolerated? Worsening of disease is obviously an adverse event to an antimicrobial agent..3. Line 635 states that mechanistically, "the energy produced by ATP hydrolysis induces a series of conformational changes which drive unwinding of RNA or DNA." A reference should be provided that best supports this precise mechanistic statement, as until recently the helicase activity was in question, as it had been inconsistently demonstrated.4. Isn't it an overstatement to say declare in the Discussion that " several comments exhibited " pan-EV and -RV activity" line 581? Pan clearly means "all" but the authors demonstrate data from only 2 of ~ 160 rhinovirus types, one-EV-B strain, and one EV-C strain (Sabin 1). Granted, at least activity is being seen against strains from 6 of 7 species...but it is an overstatement to call this pan-EV activity, and a more modest claim is appropriate. 17 Sep 2020Attachment20200828_response_to_reviewer.docxSubmitted filename: Click here for additional data file. 22 Sep 2020Dear Dr van Kuppeveld,On behalf of my colleagues and the Academic Editor, Tobias Bollenbach, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology. The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.Early VersionThe version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.PRESS biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.Kind regards,Alice MussonPublishing Editor, PLOS Biologyon behalf ofDi Jiang, PhD,Senior EditorPLOS Biology